Isothermal Titration Calorimetry has become more prevalent in biomolecular science, research and engineering. With an increasing need to characterise biomolecules and their interactions, it’s important for researchers and scientists across many fields to understand how this unique instrumentation functions.

In this article you’ll discover exactly how ITC instruments can be used to measure biomolecular interactions and how they are used in drug development and other industries.

What is Isothermal Titration Calorimetry?

ITC works by directly measuring the heat that is either released or absorbed during a biomolecular binding event.

Used in quantitative studies of biomolecular interactions, Isothermal Titration Calorimetry is the only known technique that can determine all binding parameters simultaneously in a single experiment. This fact alone makes Isothermal Titration Calorimetry an important component in biomolecular study.

ITC can also measure the affinity of binding partners in their native states, doing so without modification with fluorescent tags or immobilisation.

Common uses of Isothermal Titration Calorimetry

Traditionally, ITC is most commonly used for investigations into protein interactions, including:

• Protein-protein interactions
• Protein-DNA/RNA interactions
• Protein-small molecule interactions
• Enzyme kinetics

In the above interactions, Isothermal Titration Calorimetry provides the most direct route to a more complete thermodynamic characterisation of protein interactions.

Furthermore, over the past few years ITC has seen rapid development and usage uptake. It has gone from from a specialised method focused on understanding the interactions of molecular biological processes to newer applications in protein folding and misfolding. This works to expand the use case of ITC methodology and instrumentation.

What does ITC measure?

Isothermal Titration Calorimetry measures heat released or absorbed by complexes formed between molecules. This provides information on the affinity of binding partners in a native state. From here, operators can utilise experiment data to generate accurate information on:

• Binding constants (KD)
• Reaction stoichiometry (n)
• Enthalpy (ΔH)
• Entropy (ΔS)

This occurs in part through the introduction of a ligand into the biomolecular sample.

During a binding occurrence, heat will either be absorbed or released. Progressive titration of the ligand into a sample cell containing a biomolecule allows the sensitive calorimeter to take a measurement.

What makes Isothermal Titration Calorimetry unique?

A complete characterisation of epigenetic proteins requires a clear interaction between a specific module in isolation and its mark. Without this interaction it’s difficult to characterise the structure, function and activity of an epigenetic proteins.

What makes ITC unique is that it is the only known method for characterising all known binding parameters in a single experiment. It is therefore more efficient at producing results than most of the other instruments available in the field.

How ITC instrumentation works

The Malvern MicroCal PEAQ ITC is designed for ease-of-use with exceptional sensitivity and works along the following guidelines.

Preparing the thermal core

The microcalorimeter has two cells, one containing water, which is the reference cell. The other cell contains the sample you are testing. The calorimeter keeps these two cells at the same temperature while the heat sensing component detects any temperature difference between each of the cells when binding occurs.

This thermal core is at the heart of every Isothermal Titration Calorimetry experiment. Careful preparation according to the instrument manual is essential for a successful and accurate experiment.

Taking measurements

As stated above, ITC requires progressive titration of a ligand into the sample cell.

The ligand, which is loaded into a syringe, is inserted into the sample cell. From here, aliquots of ligand are injected into the protein sample.

If the ligand binds to the protein it will cause heat changes within the sample. The ITC instrument’s capability means even if the change is only a few millionths of a degree, it can be detected and measured.

In all cases, the quantity of heat measured is directly proportional to the amount of binding.

Analysis of data and results

The molar ratio between the ligand and protein is gradually increased through a series of ligand injections.

This causes the protein to become saturated and less binding occurs between it and the ligand. Heat exchange decreases until an excess of ligand brings about the reaction towards saturation. Once this occurs, the experiment component ends.

From here, data from the instrument can be accessed using software to produce graphs and reports.

ITC and drug development

ITC is predominantly used in drug development specific to the following areas:

• Quantification of binding affinity
• Selection and optimisation of candidates
• Thermodynamic and concentration measurements
• Mechanism of action characterisation
• To confirm intended binding targets in molecules
• Binding specificity and stoichiometry determination
• Enzyme kinetic measurements

With an ever increasing focus on biomolecular research and drug delivery, many research facilities find themselves in search of the right Isometric Titration Calorimetry instrument.

Introducing the Malvern MicroCal PEAQ ITC

When it comes to stand out ITC instrumentation, the Malvern MicroCal is an exceptional frontrunner. Delivering an accurate and sensitive analysis with minimal resource consumption, this is the ideal device for most laboratories. Discover more about this automated solution here. Or contact us today for a full quote.

Differential Scanning Calorimetry (DSC) is primarily used to characterise the stability of biomolecules like proteins. Importantly, it is a direct measurement of the biomolecule in its native form. DSC measurements can provide data on thermal stability and serve as a structural fingerprint to assess conformation.

By heating the molecule at a constant rate, measurement is made of the change in heat associated with thermal denaturation of the biomolecule.

Advantages of Differential Scanning Calorimetry

Because DSC relies on heat measurements, it’s possible to characterise native biomolecules without necessarily having optically clear samples.

Characteristics measured through DSC provide the melting temperature, but also data on folding and unfolding forces within the biomolecule.

Uses of Differential Scanning Calorimetry

DSC is used frequently in drug development. Some of the key areas in which a calorimeter might be used include:

• Selection and characterisation of stable proteins and potential candidates in biotherapeutic developments
• Interaction studies of ligands
• Optimisation of manufacturing and purification conditions
• Determination of best conditions for liquid formulation

Understanding Basic Equilibrium Thermodynamics

To understand how DSC works and the benefits it can offer for accurate biomolecular measurement, it is important to understand Basic Equilibrium Thermodynamics.

Essentially, proteins and other biomolecules can be transferred between N and D conformations:

• N – structured, native and biologically active conformation
• D – unstructured, denatured, and inactive conformation

This transference happens through an increase in temperature beyond a specific range. The process is not limited to just proteins. Other biomolecules affected include:

• Organic polymers
• Biological macromolecules like DNA and lipids

Instruments used in Differential Scanning Calorimetry

The key instrument used for DSC measurement is a calorimeter. Newer models, like the Malvern MicroCal PEAQ DSC, provide a number of automation benefits in sampling and analysis. Unattended operation of the instrument after sample loading frees operator time, increasing productivity. Automated data analysis has led to more rapid generation of highly reproducible thermal stability data and compliance with regulatory requirements.

DSC instrumentation usage and protocols

Proper usage and protocols are essential for ensuring the instrumentation and samples are correctly prepared and experiments safely executed. These protocols are also documented in the US Library of Medicine.

Starting up the instrument

General guidelines might differ from instrument to instrument. Always check the instrument manual or contact the manufacturer when in doubt.

1. Turn on the calorimeter and increase cell pressure. This suppresses sample boiling and the prevention of bubbles. In most calorimeters, the use of nitrogen is used.
2. Adjust pressure of nitrogen gas in accordance with the manufacturer’s guidelines. This should be in line with the constituting material of the cell.
3. Fill cleaning agent reservoirs to the correct volume.
4. Holding compartment temperature should be set to the right value in order to maintain sample integrity before starting the experiment.

Preparing the sample

1. Dialyse your sample against the buffer that is the reference for the experiment. As an alternative, elution buffer collected during the final step of protein purification can be used instead.
2. Use the Kjeldahl method or Lowry method to determine the concentration of the sample. Use the most suitable method. Continue to use the same method throughout a single study to maintain objective results comparison.
3. Degas both sample and buffer in a vacuum. The purpose here is to remove microbubbles that can lead to volume inaccuracy. In some newer calorimeters this can be skipped.
4. This step requires a micropipette and sterile tips in a laminar flow biocontainment cabinet. In pairs, load both the samples and their buffers into 96 well plates compatible with the calorimeter.
5. Fill the first two pairs of wells with buffer only and the last two pairs with water only. A buffer-buffer scan will help verify the instrument and establish baseline. Water scans will clean the cells.
6. Ensure well plate is properly sealed with sealing film before removing the plate from the biocontainment cabinet. This helps prevent contamination.
7. Ensure plate is in the correct orientation when placing into the sample holding compartment of the instrument.

Setup parameters for the experiment

1. Enter sample information into the software. If available, enter concentrations as well. If not, input concentration values into analysis software before data analysis.
2. Set cleaning options
3. Set starting temperature of the experiment based on the sample. If sample is unknown, start with a low temperature.
4. Set experiment final temperature. This is dependent on the sample.
5. Select the scan rate for the experiment. This is also dependant on the individual experiment, but it typically is either 60 °C/h, 90 °C/h or 120 °C/h. If you are scanning an unknown sample it’s recommended to scan at different rates and examine the kinetics of unfolding within the sample.
6. In order to determine reversibility of thermal unfolding you should rescan the samples. Unfolding is to be considered reversible if the enthalpy for the second scan is at least 80% of the enthalpy value of the first scan.
7. To preserve the cells in the calorimeter set the thermostat to 10 °C post experiment
8. Before starting the experiment, double check all parameters and ensure they meet required guidelines for the scope of the experiment.

Analysing the data with your calorimeter

Retrieval of data from the experiment can provide a number of insights into the sample. For ease of use, more modern calorimeters like the Malvern MicroCal PEAQ DSC provide a number of automated processes so you can gain faster, more accurate insights and results. Find out more about the MicroCal PEAQ DSC here.

Quartz Crystal Microbalance allows for the accurate measurement of thin films interactions by utilising the unique properties of quartz. However, it is only with the addition of dissipation measurement (QCM-D) that you can truly unlock the power of QCM instruments.

How did Quartz Crystal Microbalance come about?

As early as 1880 Jacques and Pierre Curie discovered that when mechanical stress is applied to quartz a voltage proportional to the stress is also created.

However, the term Quartz Crystal Microbalance wasn’t coined until the late 1950s by Sauerbrey, who also demonstrated that the oscillation frequency of the quartz was dependant on changes in surface mass. This led to the discovery of the Sauerbrey equation.

The Sauerbrey equation shows that when voltage is applied to a quartz crystal to produce a specific oscillation, any change in the mass of the quartz surface is directly related to the change in frequency of the oscillating crystal.

Where is the Sauerbrey equation applicable?

The Sauerbrey equation says that the change in QCM frequency determines the mass of the adsorbed analyte. It is valid for elastic or rigid samples like:

• Metal oxides
• Metallic coatings
• Thin adsorbed layers

As these materials do not dissipate energy during oscillation. The equation relies on a linear sensitivity factor that is fundamental to the properties of the quartz, so in theory it doesn’t require calibration.

However, this is only applicable to uniform thin film samples. That’s why the Sauerbrey equation is not valid with inelastic materials like:

• Polymers
• Biomolecular systems
• Cells

As viscous dampening during oscillation causes energy loss, soft and viscoelastic films fail to adequately couple with the oscillating quartz crystal. This can lead to an underestimation of the mass using the Sauerbrey relationship.

To fully characterise these types of materials QCM-D (Quartz Crystal Microbalance with Dissipation) was created. Today, QCM-D instruments are among the leading tools for measuring thin film interactions (mass, thickness, density) and viscosity. Additionally, associated solvent or water content of adsorbed films can be measured by comparing the mass measured using QCM-D with that of complementary techniques such as ellipsometry or surface plasmon resonance.

What is a Quartz Crystal Microbalance?

A QCM consists of a thin disk made from quartz and sandwiched between coated electrodes. AC voltage oscillates the sensors. As the sample material flows across the sensors, the mass changes and so does the frequency (Sauerbrey equation).

The QCM acoustic waves must propagate perpendicular to the surface of the crystal. Therefore, in the manufacturing process there is a specific method where the crystal axes are cut in either:

• AT
• BT

With AT the most common method of manufacturing the QCM as it provides for almost zero temperature dependency.

Resonance sensitivity

The thickness or thinness of the quartz crystal determines the sensitivity to resonance. The thinner the QCM, the higher the resonant frequency. Conversely, thinner quartz is also more fragile.

Common uses of Quartz Crystal Microbalance

Though the technology has existed for over half a century, it is mainly in the last few decades that QCMs have seen a rapid uptake in adoption. Uses for Quartz Crystal Microbalance include both research and industrial application:

• Surfactant adsorption on surfaces
• Contamination monitors
• Chemical analytes detection
• Environmental pollutant measurements
• Biomolecule.and disease biomarker measurements
• Pathogen detection
• Hydrogen adsorption on metal films
• Metal detection in vacuum
• Electrovalency measurements
• DNA and RNA hybridisation studies
• Gas chromatography detection
• Antigen-antibody reactions

Advantages over other instrumentation

In the area of chemical detection analysis is typically carried out by the following instruments/techniques:

• FTIR – fourier transform infrared spectronomy
• GC – gas chromatography
• MS – mass spectrometry

All of these techniques can be cumbersome and expensive, not to mention time consuming and limited to offline analysis.

QCM offers several advantages over these techniques:

• Low overheads
• Rapid results
• Safe, real time procedure
• Portability

Biolin Q-Sense Analyser

The Q-Sense Analyzer by Biolin represents the ideal qualities of a QCM. Features include:

• Real time analysis of thin films
• QCM-D technology (Quartz Crystal Microbalance with Dissipation)
• Measurement across two parameters:
  • Frequency – Mass / thickness
  • Dissipation – Rigidity
• 4 flow modules that can be used serially or in parallel
• The ability to measure samples of thin films, polymers, surfactants, proteins, lipids and cells

Integrated software

Q-Sense Dfind software provides a single suite of integrated analysis tools especially designed to interface with the Q-Sense Analyser.

Dfind is an intuitive software experience that first provides the user with the raw data and then guides them through modelling and reporting structures on elements like:

• Mass
• Thickness
• Viscoelastic properties
• Adsorption rate

QCM-D

Unlike many QCM systems the Biolin Q-Sense Analyser contains dissipation measurement to provide data on structure and viscoelasticity of the sample. Without the dissipation measurement QCM’s can only rely on the Sauerbrey equation which severely hinders the capability of the instrument.

Quartz Crystal Microbalance with Dissipation, QCM-D, provides real-time, label free measurements of molecular adsorption and interactions on various surfaces with nanogram sensitivity. In addition to adsorbed mass, measured as changes in frequency of the quartz crystal, the dissipation parameter (D) provides novel insights regarding structural (viscoelastic) properties of adsorbed layers.

The best in scientific instrumentation

ATA Scientific is committed to sourcing and supplying the best in scientific instruments. The Biolin Q-Sense Analyser is one of many top-of-the-line instruments for precision lab and industrial sampling. Contact ATA Scientific today for a competitive quote on your next instrument purchase, and find out why we’re the specialists in analytical instrumentation.